Interleukin-1 (IL-1), a major pro-inflammatory cytokine, has a wide range of biological activities in inflammation. The IL-1 receptor belongs to the IL-1R (IL-1 receptor)/TLR (Toll-like receptor) superfamily. Human TLRs have recently emerged as key components in the generation of immune and inflammatory responses due to their ability to recognize pathogen associated molecules. Much effort has been devoted towards the understanding of molecular mechanisms by which the IL-1R/TLRs mediate signaling, with the long-term objective to develop more effective anti-inflammatory small molecule drugs. While we are interested in the IL-1R/TLR signaling in general, this application focuses on the IL-1- mediated pathway, since the IL-1 pathway is likely to provide a "prototype" for the similar yet distinct pathways mediated by TLRs. Genetic and biochemical studies revealed that IL-1R mediates a very complex pathway, involving a cascade of kinases organized by multiple adapter molecules into signaling complexes, leading to activation of the transcription factor NFkappaB. Based on studies by our group and others, we have postulated a model for the IL- 1 pathway. Upon IL-1 stimulation, the IL-1 receptor mediates the formation of Complex 1, where IRAK4 (IL-1 receptor-associated kinase 4) is activated, leading to hyperphosphorylation of IRAK, which creates an interface for its interaction with adapter Pellino 1. The receptor proximal components are then released from the receptor to form an Intermediate Complex, followed by formation of Complex II, leading to phosphorylation of TAK1 (TGFbeta activated kinase, a MAP3K) and TAB2 (TAK1 binding protein 2) on the membrane. Complex III is then dissociated from Complex II and translocated from the membrane to the cytosol, where TAK1 is activated, followed by the activation of IKK (IkappaB kinase) and NFkappaB. While this model is well supported by the published studies and our preliminary data, the detailed molecular mechanisms for our model are largely lacking. The goal of this proposal is to elucidate the detailed molecular mechanisms for IL-1-induced receptor proximal signaling events, including formation and activation of the receptor complex (Aim 1), release of signaling components from the receptor (Aim 2), and translocation and activation of TAK1 (Aim 3).